1. Field of the Invention
This invention relates to a method for measurement of a trace enzyme, more particularly, to a method for photochemical measurement of a trace enzyme.
2. Development of the Invention
Various methods have heretofore been developed for measuring the activity of a trace enzyme. For example, there are: a turbidimetric method wherein the decrease in turbidity caused by an enzyme reaction is traced using a suspension of a high molecular weight substrate; an absorptiometric method wherein a high molecular weight substrate is decomposed or cleaved by an enzyme and, after precipitating and removing undecomposed substrate, soluble component is determined by an absorbance measurement; a method wherein a dye or fluorescent substance is previously bound to a high molecular weight substrate, an enzymatic reaction is effected to decrease the molecular weight of the dye or fluorescent substance, and the fractionated dye or fluorescent substance of lower molecular weight is measured; and a method of quantitative assay wherein, using a substrate which is designed to change in absorption spectrum, form a color or form a fluorescent substance, based on a splitting-off or change in a part of the substrate after an enzymatic reaction, the resulting absorbancy or fluorescent intensity is measured, etc. (SEIKAGAKU JIKKEN KOZA (lectures on Biochemical Experiments), vol. 5, subtitled "Study on Enzymes", edited by the Biochemical Association, Japan, published by Tokyo Kagaku Dojin, 1975).
Most of these methods, however, quantitatively determine an amount of enzyme on the order of .mu.g/ml. Even utilizing a type of substrate releasing a fluorescent substance (e.g., derivatives of coumarin, umbelliferone, etc.), which is recognized to be most sensitive among these conventional methods, it is only possible to measure an enzyme quantity on the order of ng/ml.
Therefore, in activity measurements of trace enzymes labelled in accordance with enzyme immunoassay, the development of more stable and more highly sensitive enzyme activity measurement methods has been desired.
Further, since the enzymes as biocomponents in blood, body fluids, urine and in tissues in the living body such as various organs, the brain, etc. mostly are present in a very small quantity, except for certain enzymes (amylase, GOT, GPT, etc.) which exist in a large amount, such enzymes cannot be determined by conventional measurement methods. Therefore, radioimmunoassay (hereinafter merely "RIA") which is an immunological measurement method using a radioactive isotope has recently been introduced. The principle of RIA is described in, for example, Kumahara and Shizume, RADIOIMMUNOASSAY, New Edition, pages 3 to 10 (1977), published by Asakura Publishing Co., Ltd., KISO SEIKAGAKU JIKKENHO (Basic Biochemical Experiments) (6), Biochemial Assay (1967), published by Maruzen Co., Ltd., Tokyo, METHODS IN ENZYMOLOGY, edited by Sidney P. Colowick et al, vols. I, II, III, V and VII, published by Academic Press, New York and The Enzyme, vols. 3, 4 and 5, Paul D. Boyer et al (1971), published by Academic Press, New York.
However, RIA as a quantitative assay for enzymes has disadvantages such as: (1) since it is an immunoassay, there is the possibility that the activity of an enzyme--which is the functional characteristic of the enzyme--will not be actually reflected; (2) there is the possibility that analogous enzymes and precursors having a similar antigenic site might be included in analytical data; and (3) in the case where the enzyme to be measured, for example, such as an enzyme in an antigen or antibody labelled with the enzyme used for the enzyme immunoassay, is bound to another component but not present in the free state, it is difficult to prepare an antibody corresponding to the aforesaid labelled antigen or antibody and design for a method for measurement is practically difficult.
RIA has further disadvantages due to the use of radioisotopes. That is: (1) potential injury to the person dealing with radiation is a matter of concern; (2) special places and controls are required for storage and waste disposal of the radioactive substances used; (3) the amount of radiation from the radioisotope is reduced with the passage of time due to the half decay of the isotope; and (4) measurement of the count of radioactivity requires expensive devices.
The information obtained by measuring enzyme activity using a specific substrate under given reaction conditions (e.g., concentration of substrate, total volume, reaction pH, reaction temperature, reaction time, ionic strength, salts co-existant, etc.) is generally classified as follows:
(1) The sum of enzyme activities having a catalytic action on the structure of the substrate in the system; or
(2) Under the condition of constant enzyme concentration, a degree of inactivation depending upon purity of an enzyme, the presence or absence of inhibitors, intensity of inhibition, denaturation, etc., i.e., measure of specific activity.
Finally, the purpose of measuring the activity of an enzyme as a component in the living body is frequently to obtain mainly information per (1) above and the purpose of activity measurements of enzyme-labelled materials exterior a living body is to obtain mainly information per (1) and (2) above. As is well known, in any case, specific enzyme activity can only be measured by selecting a substrate corresponding to enzyme specificity.
The term "specificity" referred to herein is art-recognized and defines selective reactivity between substances, e.g., of an enzyme with its corresponding substrate.